Elisaveta Milusheva

Regulatory interactions among axon terminals affecting the release of different transmitters from rat striatal slices under hypoxic and hypoglycemic conditions

Abstract: An in vitro model of ischemia was utilized to study the effects of both oxygen and glucose depletion on transmitter release from rat striatal slices. The spontaneous and stimulation‐evoked releases of tritiated dopamine, γ‐aminobutyric acid, glutamate, and acetylcholine were measured. Hypoxia increased the evoked release of glutamate and dopamine without effect on the resting release. In contrast, hypoglycemia itself increased the resting release of dopamine. Hypoxia in combination with hypoglycemia provoked a massive release of glutamate, dopamine, and γ‐aminobutyric acid. The effect on acetylcholine release was less pronounced. Ca2+ withdrawal partly reduced the effect of hypoxia combined with hypoglycemia on dopamine release and application of tetrodotoxin (1 μM) abolished it. MK‐801 (3 μM), an N‐methyl‐d‐aspartate receptor antagonist, attenuated the effect of hypoxia and hypoglycemia on [3H]dopamine release. ω‐Conotoxin (0.1 μM) had a similar effect on stimulation‐evoked release under a hypoxic condition. The D2 receptor antagonist sulpiride (100 μM) failed to enhance the release of [3H]acetylcholine in hypoxia combined with hypoglycemia. It was suggested that in response to hypoxia combined with hypoglycemia there is a massive release of glutamate due to the increased firing rate which in turn releases dopamine from the axon terminals through stimulation of presynaptic N‐methyl‐d‐aspartate receptors. Dopaminergic inhibitory control on ACh release seems not to be operative under conditions of hypoxia combined with hypoglycemia.

Inhibitory effect of hypoxic condition on acetylcholine release is partly due to the effect of adenosine released from the tissue

Isolated longitudinal muscle strip with Auerbachs plexus attached was used to study the stimulation-evoked release of 3H-acetylcholine (3H-ACh) under normoxic and hypoxic conditions. Hypoxia reduced the release of ACh. Theophylline, a purinoceptor P1 antagonist and vinpocetine, an antiischemic compound partly reversed the effect of hypoxia. Unlike theophylline, the effect of vinpocetine was not mediated via adenosine action, since it failed to affect the presynaptic action of adenosine, and the effect of theophylline and vinpocetine was additive. When they were added together the effect of hypoxia was almost completely antagonized. Dipyridamole, an adenosine uptake inhibitor, potentiated the effect of hypoxia and the presynaptic inhibitory action of adenosine on ACh release. Evidence was obtained that the effect of hypoxia is at least partly due to adenosine formed from purine nucleotides.

Excessive release of [3H]noradrenaline and glutamate in response to simulation of ischemic conditions in rat spinal cord slice preparation: effect of NMDA and AMPA receptor antagonists

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.

Non-synaptic release of [3H]noradrenaline in response to oxidative stress combined with mitochondrial dysfunction in rat hippocampal slices.

Brain ischemia is frequently associated with oxidative stress in the reperfusion period. It is known that noradrenaline (NA) is released in excess under energy deprivation by the sodium-dependent reversal of the monoamine carrier. However, it is not known how oxidative stress affects NA release in the brain alone or in combination with energy deprivation. As a model of oxidative stress, the effect of H(2)O(2) (0.1-1.5 mM) perfusion was investigated in superfused rat hippocampal slices. It elicited a dose-dependent elevation of the release of [(3)H]NA and its tritiated metabolites as well as a simultaneous drop in the tissue energy charge. Mitochondrial inhibitors, i.e. rotenone (10 microM), and oligomycin (10 microM) in combination, also decreased the energy charge, but they had only a mild effect on [(3)H]NA release. However, when H(2)O(2) was added together with oligomycin and rotenone their effect on [(3)H]NA release was greatly exacerbated. H(2)O(2) and mitochondrial inhibitors also induced an increase in [Na(+)](i) in isolated nerve terminals, and their effect was additive. The effect of H(2)O(2) on tritium release was temperature-dependent. It was also attenuated by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM) and (+/-)-2-amino-5-phosphonopentanoic acid (10 microM), by the nitric oxide synthase inhibitors, N omega-nitro-L-arginine methyl ester (100 microM), or 7-nitroindazole (50 microM) and by the vesicular uptake inhibitor tetrabenazine (1 microM). Our results suggest that oxidative stress releases glutamate followed by activation of postsynaptic ionotropic glutamate receptors that trigger nitric oxide production and results in a flood of NA from cytoplasmic stores. The massive elevation of extracellular NA under conditions of oxidative stress combined with mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus initiating a self-amplifying cycle leading to neuronal degeneration.

The effect of antiparkinsonian drugs on oxidative stress induced pathological [3H]dopamine efflux after in vitro rotenone exposure in rat striatal slices.

An in vitro model of mitochondrial dysfunction with subsequent oxidative stress was elaborated and utilized to study the effect of drugs, currently used for the treatment of Parkinsons disease, on pathological H(2)O(2)-evoked [(3)H]dopamine efflux and the formation of toxic dopamine metabolites in rat striatal slices. 60 min rotenone (0.1-10 muM) pretreatment decreased dopamine content and [(3)H]dopamine uptake, as well as ATP level and energy charge of the slices. In addition, a robust potentiation of H(2)O(2)-evoked [(3)H]dopamine efflux and the formation of dopamine quinone in the effluent was detected. l-DOPA (200 muM) markedly elevated resting but not 100 muM H(2)O(2)-evoked and electrically-induced [(3)H]dopamine efflux. Furthermore, l-DOPA promoted the formation of dopamine quinone. Ropinirole (100 nM) did not affect resting and H(2)O(2)-evoked [(3)H]dopamine efflux and inhibited the electrically evoked release only in untreated slices. l-deprenyl, at concentration of 0.01 muM potentiated, whilst between 1 and 50 muM diminished H(2)O(2)-evoked [(3)H]dopamine efflux. Rasagiline (0.01-50 muM) slightly inhibited H(2)O(2)-evoked [(3)H]dopamine efflux, and it was able to prevent the generation of dopamine quinone. Neither of the drugs was able to suppress both the pathological H(2)O(2)-evoked [(3)H]dopamine efflux and the formation of dopamine quinone with simultaneous augmentation of electrically evoked [(3)H]dopamine release what should be a future concept of antiparkinsonian drug-design.

Muscarinic modulation of nitrergic neurotransmission in guinea-pig gastric fundus.

Abstract  Muscarinic receptor activation by (4‐Hydroxy‐2‐butynyl)‐1‐trimethylammonium‐m‐chlorocarbanilate chloride (McN‐A‐343) was investigated both on NADPH‐d staining and on electrically induced responses in guinea‐pig gastric fundus. McN‐A‐343 (10 μmol L−1) significantly increased the optical density of NADPH‐d positive neurones, while blockade of nitric oxide synthase with Nω‐nitro‐L‐arginine (L‐NA) decreased it, suggesting facilitation of nitric oxide (NO) production. Electrical field stimulation (EFS; 2 Hz, 0.2 ms, supramaximal current intensity, 10 s train duration) elicited on‐contraction followed by off‐relaxation in the circular muscle strips. McN‐A‐343 (10 μmol L−1) transformed the EFS‐evoked response from on‐contraction into on‐relaxation, which was neurogenic, tetrodotoxin‐sensitive and hexamethonium‐resistant. L‐NA partly reduced the EFS‐evoked relaxation, revealing two components: a nitrergic and a non‐nitrergic one. The effect of McN‐A‐343 on the amplitude of the EFS‐evoked relaxation was not changed by the M3 receptor antagonist para‐fluoro‐hexahydro‐sila‐difenidol hydrochloride, but was significantly enhanced by M1 receptor blockade with telenzepine. In the presence of telenzepine, the L‐NA‐dependent nitrergic component of the EFS‐induced relaxation predominates. We suggest that cholinergic receptor activation has a dual effect on nitrergic neurotransmission: (i) stimulation of NOS by muscarinic receptor(s) different from M1 and M3 subtype, (ii) prejunctional inhibition of NO‐mediated relaxation via M1 receptors. In addition, M1 receptors may facilitate the non‐nitrergic relaxation.

Implication of ionotropic glutamate receptors in the release of noradrenaline in hippocampal CA1 and CA3 subregions under oxygen and glucose deprivation

A strong linkage between adrenergic and glutamatergic systems exists in the CNS but it is still unclear whether the excessive release of noradrenaline under ischemic conditions is modulated by excitatory amino acids. We studied the effect of selective glutamate receptor antagonists on the release of [3H]noradrenaline evoked by glucose and oxygen deprivation in hippocampal CA1, CA3 and dentate gyrus subregions. The release of glutamate, aspartate and GABA was measured by HPLC. Omission of oxygen and glucose increased the release of [3H]noradrenaline as well as the release of amino acids. Maximum effect on noradrenaline release was observed in CA1 region. The relative increase of the release after 30 min energy deprivation (R(2)) versus the basal release under normal conditions (R(1)), i.e. the R(2)/R(1) ratio was 7.1+/-1.0, 3.87+/-0.4 and 3.26+/-0.27 for CA1, CA3 and dentate gyrus, respectively. The [3H]noradrenaline outflow in response to glucose and oxygen deprivation was abolished at low temperature, but not by Ca(2+) removal, suggesting a cytoplasmic release process. In CA1 and CA3 [3H]noradrenaline release was significantly attenuated by MK-801, an NMDA receptor antagonist. The AMPA receptor antagonist GYKI-53784 had no effect in CA3, but partly reduced noradrenaline release in CA1. Our results suggest that ionotropic glutamate receptors seem to be implicated in the massive cytoplasmic release of noradrenaline in CA1 what may contribute to its selective vulnerability.

Glutamate stimulation of acetylcholine release from myenteric plexus is mediated by endogenous nitric oxide.

Glutamate was found to be an excitatory neurotransmitter in the enteric nervous system. Although several lines of evidence indicate a role of glutamate in the regulation of gut motility and secretion the physiological significance of glutamatergic transmission is not clear yet. We studied the effect of glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining in longitudinal muscle strips with attached myenteric plexus of guinea pig ileum. L-glutamate (100 microM) significantly enhanced both the evoked [3H]acetylcholine release and the optical density of nicotinamide adenine dinucleotide phosphate-diaphorase positive neurones, i.e. the intensity of staining. The non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (3 microM) abolished the stimulatory effect of L-glutamate on acetylcholine efflux. Similarly, the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (100 microM) significantly reduced the effect of L-glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining. Our data suggest that endogenous nitric oxide seems to mediate the stimulatory effect of glutamate on acetylcholine release from guinea pig myenteric neurons.

Modulation of dopaminergic neurotransmission in rat striatum upon in vitro and in vivo diclofenac treatment.

Diclofenac (DCF) is a widely used non‐steroidal anti‐inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10–25 μmol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)‐evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2‐evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]β‐phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre‐treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction.

Neurochemical changes in animal models of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative syndrome associated with selective loss of dopaminergic neurons in the striatum. It is believed that both mitochondrial dysfunction and oxidative stress (OS) play important role in the pathogenesis of PD. Development of an effective causal therapy should be focused on preventing or at least retarding the neurodegenerative process underlying the disease. In chronic in fusion PD model studied the effect of rotenone on the content, and release of striatal neurotransmitters of rat upon OS by H2O 2 perfusion. The effects of anti-Parkinsonian drugs were examined in vitro rotenone PD, OS slices model. The role of P2X7 receptor (P2X7r) formed by 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) or in vitro rotenone treatment using C57/BI6 and P2X7r knockout mice. Neurochemical response was determined using microvolume perfusion method, HPLC separation of dansylated samples. Dopamine o-quinone (DAQ) oxidized metabolite of DA was detected electrochemically at -100 mV potential in reduction working mode. The dopamine content in the rat striatum is decreased in response to chronic intravenous rotenone infusion. However, surviving dopaminergic neurons take up and release only a slightly lower amount of DA in response to electrical stimulation. Striatal dopaminergic neurons showed increased susceptibility to OS, responding with enhanced release of DA and with formation of toxic metabolite DAQ. The loss of DA and ATP and formation of DAQ induced by OS confirmed the impairment of striatum upon in vitro rotenone. Formation of DAQ at the end effect of OS confirmed the impairment of dopaminergic function by rotenone. However, when applied rotenone with l-DOPA treatment the endogenous DAQ was detected in perfusion samples. However, when applied rotenone with l-DOPA treatment the endogenous DAQ was detected in the micro-volume perfusion samples. Conversely, when rasagiline and trolox were in use these DA-quinones metabolites were not detectable. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7 deficient mice. Although the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors. Genetic deletion of P2X7 receptors did not change depletion of striatal endogenous DA content after in vivo MPTP or in vitro rotenone treatment. Although the endogenous amino acids content and the level of the endocannabinoid, AEA was elevated by MPTP in the striatum of mice, but 2-AG remained unchanged in mice deficient in P2X7 receptors. In conclusion, oxidative stress induced, pathological DA release and the formation of toxic metabolites of DA are distinctly modified by the tested antiparkinsonian drugs and P2X7 receptor deficiency does not promote the survival of dopaminergic neurons.